Patent application title: Novel cutinases, their production and uses
Antti Nyyssola (Espoo, FI)
Hanna Kontkanen (Espoo, FI)
Mari Hakkinen (Espoo, FI)
Ville Pihlajaniemi (Espoo, FI)
Markku Saloheimo (Espoo, FI)
Johanna Buchert (Espoo, FI)
Tiina Nakari-Setala (Espoo, FI)
TEKNOLOGIAN TUTKIMUSKESKUS VTT
IPC8 Class: AC12N918FI
Class name: Micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition preparing oxygen-containing organic compound carboxylic acid ester
Publication date: 2014-08-21
Patent application number: 20140234921
The present invention relates to novel polypeptides, or fragments of
polypeptides, genes encoding them and means for producing said
polypeptides. In detail the invention relates to polypeptides having
esterase, suberinase and/or cutinase activity at low pH. This invention
relates also to compositions containing the polypeptides and methods of
using the polypeptides.
1. A polypeptide comprising an amino acid sequence having at least 70%
sequence identity to SEQ ID NO: 1 or fragment of said polypeptide.
2. The polypeptide or fragment of claim 1, further comprising at least one esterase activity.
3. The polypeptide or fragment of claim 2, wherein the activity is a cutinase activity, a suberinase activity or a lipase activity, or any combination of said activities.
4. The polypeptide or a fragment of claim 2, further comprising a cutinase, a suberinase and a lipase activity.
5. The polypeptide or a fragment of claim 1, wherein the polypeptide or the fragment is active towards polyesters at pH range from 2.5 to 7.5.
6. The polypeptide or a fragment of claim 1, wherein the polypeptide or the fragment is active towards polyesters at pH below 5.
7. The polypeptide or a fragment of claim 1, further comprising at least 77 amino acids.
8. The polypeptide or the fragment of claim 1, wherein protein is derived from Sirococcus.
13. An enzyme preparation comprising a polypeptide having an ammo acid sequence having at least 70% sequence identity to SEQ ID NO: 1 or fragment of said polypeptide.
14. A method of hydrolysis comprising the steps of: contacting a material containing ester bonding with a polypeptide or a fragment of the polypeptide, said polypeptide having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, under conditions suitable for hydrolysis.
15. Method of claim 14, further comprising treating agricultural or food raw materials or by-products obtained from vegetables, fruits, grapes, berries or cereals.
16. Method of claim 14, further comprising treating wood raw materials, pulp and paper products, or process wastes or waters, or by-products with said protein.
17. Method of claim 14, further comprising modifying synthetic or other man-made polyester fibres or textiles with said protein.
18. Method of claim 14, further comprising removing stickies or fat from laundry and dishes with said protein.
19. Method of trans- or interesterification comprising the steps of: contacting a material to be esterified with a polypeptide or a fragment of the polypeptide, said polypeptide having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, under conditions suitable for esterification.
FIELD OF THE INVENTION
 This invention relates to novel polypeptides, particularly to polypeptides having cutinase activity also in acidic pH. The invention also relates to recombinant polynucleotides, vectors and host cells that are usable in producing said polypeptide, to method of obtaining said polypeptide and compositions comprising the polypeptide. Further, the invention relates to methods of hydrolysis of ester bonding and uses of the polypeptide.
DESCRIPTION OF RELATED ART
 Cutin is an abundant biopolymer present in the protective, waxy layer on the surfaces of aerial parts of plants. It is composed of variably substituted fatty acids interlinked with ester bonds. The typical structural components of cutin are hydroxyl and epoxy substituted ω-hydroxy fatty acids with C16 and C18 carbon chains. The cutin polymer is formed via esterification of primary alcohols. Suberin is another common protective biopolymer. It is found for example in tree bark, cork and in roots. Suberin is composed of a polyphenolic domain and a polyester domain. The polyester domain is chemically similar to cutin, but may differ somewhat in its fatty acid composition (fatty acids with longer carbon chains may be present) and in the form of cross-linking Glycerol is a major suberin component in some species and acts as the cross-linker between the suberin monomers.
 Cutinases are enzymes, which catalyze the hydrolysis of the ester bonds of cutin, suberin, lipids, waxes and other esters. All of the biochemically well-characterized cutinases are serine esterases, containing the Ser-His-Asp triad similar to serine proteases and several lipases (Carvalho et al., 1998). All known cutinases have a neutral or alkaline pH-optimum, with low or negligible activity at low pH. The cutinases that have been reported to be functional at slightly acidic pH include the following. In a report by Shishiyama et al. (1970) a cutinase from Botrytis cinerea was shown to have optimum activity at pH 5 with no detectable activity at pH 4.0 and below. Salinas et al. (1986) describe a Botrytis cinerea cutinase with a pH optimum between 5.5 and 6.0. However, no data is given on the activity of the enzyme at lower pH. In another report on a Botrytis cinerea cutinase a pH optimum of 5.6 was determined, but no information on the activity at other pH-values was given (van der Vlugt-Bergmans et al., 1997). A Trichoderma reesei cutinase has been reported to have a local pH optimum at pH 4 for p-nitrophenylbutyrate (pNPB) esterase activity. The effect of pH on the hydrolytic activity towards the natural substrates, cutin or suberin, were however not reported for this enzyme (WO2009007510). Trail and Koller (1993) describe two cutinases from the fungus Alternaria brassicicola, which have pH optima at 6.5 and at 8.5. These enzymes show only modest activity at pH 5.0. Koller and Parker (1989) describe a cutinase from Venturia inaequalis showing a pH optimum between pH 6.0 and 7.0. However, no data is given by the authors on the activity of this enzyme at pHs below pH 5.0.
 Cutinases have been suggested for a number of uses of which only few are mentioned here. Cutinases could be used in detergents for dishwashing and laundry applications. Cutinases having an alkaline pH-optimum are suitable for use in alkaline detergents. Cutinases having an acidic to neutral pH-optimum could be suitable for rinse conditioners, for light duty products and for industrial cleaning products (WO9403578).
 For use in the textile industry, a bioscouring method utilizing cutinases for the removal of the waxy layer present in cotton has been developed (Agrawal, 2005). Cutinases have also been used for modification of the surfaces of polyester fibers (US2002007518) and in antifelting of wool-fabrics (CN101565902).
 In addition to hydrolytic reactions cutinases can be utilized for catalyzing inter- and transesterification reactions as well as synthesis of esters (Pio and Macedo, 2009; Pinto-Sousa et al., 1994; de Barros et al., 2009). The use of cutinases for the detoxification of feed products contaminated by the heat-stable mycotoxin zearalenone has recently been patented (US2009162480). Cutinases have also been suggested for use in combination with other hydrolytic enzymes for the degradation of plant materials (US20090325240) and for pre-treating wood-containing material (WO2009042622).
 Large amounts of cutin and suberin are present in low value waste materials produced by food, agriculture and forest industries. These waxy materials are hydrophobic and their structure is recalcitrant. They may thus impair the industrial processing of plant materials.
 The use of polyesterases could improve the processing and exploitation of several plant materials, such as cereals, fruits, vegetables and berries, and also improve the release and recovery of valuable bioactive and functional components from these materials.
 Cutin is for example present in the processing waste of fruits, vegetables and berries. Furthermore, the forest industry produces massive amounts of bark waste, of which suberin is a major component. These polyester materials provide a rich source of high value chemicals, which have potential use as raw materials for example in the production of lubricants and binders.
 Many of these polyester containing materials described above are acidic. Despite their wide potential, applicability of the known cutinases is limited, since their reported cutinolytic and suberinolytic activities are either poor or nonexistent at low pH. That is, acidic materials cannot be hydrolyzed with the known cutinases. Furthermore, the pH range at which known cutinases are active is typically narrow. The known cutinases can thus be used only under restricted conditions, which is a remarkable disadvantage in industrial processes, especially when use of a mixture of enzymes is required. There is a need for cutinases that are stabile and active over a broad pH range from acidic to neutral. The present invention meets this need.
OBJECTS AND SUMMARY OF THE INVENTION
 The aim of the present invention is to provide novel polypeptides or fragments of polypeptides. Particularly the aim of this invention is to provide polypeptides having esterase, and preferably cutinase, suberinase and/or lipase activity at acidic pH and polypeptides that are usable over a wide range of pHs. Further, the aim is to provide nucleotides encoding said polypeptides, means for production of said polypeptides and preparations containing polypeptides.
 First aspect of the invention is a novel polypeptide. Characteristic to the polypeptide is that it comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 or fragment of said polypeptide.
 The second aspect of the invention is a recombinant polynucleotide. Characteristic to the polynucleotide is that it comprises a nucleotide sequence having at least 80% identity to nucleotides from 52 to 606 of SEQ ID NO: 2 or to nucleotides from 7 to 561 of SEQ ID NO: 3, or a complementary strand thereof, or a codon optimized sequence of SEQ ID NO: 2.
 The third aspect of the invention is a vector. Characteristic to the vector is that it comprises the polynucleotide of the invention.
 The fourth aspect of the invention is a host cell. Characteristic to the host cell is that it has been transformed with the vector of the invention.
 The fifth aspect of the invention is a method for obtaining the polypeptide of the invention. According to the invention the method comprises transforming a microorganism with a vector of this invention, culturing the transformed microorganism under conditions allowing the expression of said polynucleotide, and recovering the expressed polypeptide.
 The sixth aspect of the invention is an enzyme preparation. Characteristic to the enzyme preparation is that it comprises the polypeptide or a fragment thereof according to the invention.
 The seventh aspect of the invention is a method of hydrolysis of ester bonds. According to the invention the method comprises contacting material containing ester bonds with the said polypeptide or a fragment thereof according to this invention under conditions suitable for hydrolysis.
 The eight aspect of this invention is a method of trans- or interesterification. According to the invention the method comprises contacting material to be esterified with the polypeptide or the fragment of this invention under conditions suitable for esterification.
 Still the ninth aspect of this invention is a use of the polypeptide or a fragment of this invention, or enzyme preparation of this invention in food industry, pulp and paper industry, detoxification applications, textile industry, or in laundry and dishwashing applications, or in chemical syntheses.
 Preferred embodiments and advantages of the invention are described in the following detailed description with reference to the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1. Alignment of the Sirococcus conigenus cutinase amino acid sequcence with other fungal cutinase sequences. Abbreviations: Scsc--Sclerotinia sclerotiorum (GenBank XP--001558272), Boci--Botrytis cinerea (GenBank XP--001558272), Mofr--Monilinia fructicola (Uniprot Q8TGB8), ScCut--Sirococcus conigenus, Fuso--Fusarium solani f. sp. pisi (GenBank P00590). Identical residues are framed in black while similar residues are framed in grey. The three amino-acids (Ser, His, Asp) belonging to the catalytic triad are marked with rectangles.
 FIG. 2. pNPB-esterase activity (at pH 3.5) of the growth medium of the Pichia pastoris transformant expressing the gene for ScCut.
 FIG. 3. The esterase (as hydrolysis ofpNPB) and cutin hydrolyzing efficiency of ScCut. The esterase activities were determined kinetically at 25° C. The amounts of released cutin mono- and oligomers were determined at 40° C. for 24 h and are represented as percents of the maximum. The error bars represent standard deviations.
 FIG. 4. Residual pNPB-esterase activity of ScCut after 24 h of incubation at 40° C.
 FIG. 5. Effect of fatty acid chain length on the esterolytic activity of ScCut cutinase determined at 25° C. and at pH 4.5.
DETAILED DESCRIPTION OF THE INVENTION
 The invention provides novel polypeptides having a SEQ ID NO: 1 or a sequence showing at least 70% identity to SEQ ID NO:1.
 In one embodiment of the invention the polypeptide comprises an amino acid sequence that has at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 1 or fragment of said polypeptide. In further embodiment the polypeptide consists of an amino acid sequence that has at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to SEQ ID NO: 1 or fragment thereof.
 As used herein the term "identity" means the sequence identity between two amino acid or nucleotide sequences compared to each other. Particularly the identity means a global identity of respective fragments of the sequences. It is evident to a skilled man that e.g. a mature polypeptide is not comparable to a polypeptide with a signal peptide. The identity of the sequences is determined using ClustalW alignment (e.g. in using default settings and Blosum62 as the substitution matrix (Thompson et al., 1994).
 In this connection term "fragment" means a part of SEQ ID NO: 1 being able to express at least one of the activities of the mature polypeptide. Preferably the activity of the fragment is at least 30% of the activity of the mature protein. In one embodiment the active fragment has at least part of any of the activities of the polypeptide as defined in SEQ ID NO: 1, preferably it has activity of at least 40%, more preferably 50%, 60% 70% 80%, 90% or even essentially the same activity as the mature protein (i.e. amino acids 18-202 of SEQ ID NO: 1, encoded by nucleotides 52-606 of SEQ ID NO: 2 and encoded by nucleotides 7-561 of SEQ ID NO: 3). In one embodiment the active fragment comprises at least 73 amino acids, preferably amino acids 116-188 of SEQ ID NO: 1. In this embodiment the identity to the fragment is at least 76%, preferably at least 80%, more preferably at least 85%, 90%, 95% or even 98%.
 Particularly the polypeptides of this invention are esterase enzymes, which belong to the subclass EC 3.1.1 (Nomenclature of the International Union of Biochemistry and Molecular Biology) and which hydrolyze carboxylic acid esters with the formation of an alcohol and a carboxylic acid anion. Typically the enzymes of this invention have activity towards several substrates.
 Polyesters are polymers which contain an ester functional group. Polyesters may be synthetic (such as for example polyethylene terephthalate, polycaprolactone and polylactic acid) or of natural origin (such as for example suberin and cutin).
 Cutinases (cutin hydrolases, EC 184.108.40.206) are serine esterases containing typically the Ser, H is, Asp triad of serine hydrolases and catalyzing the hydrolysis of carboxylic ester bonds of cutin and suberin, but also of lipids, waxes and other polyesters and esters. Suberinase is an enzyme catalyzing the degradation of suberin. Lipases (triacylglycerol lipases EC 220.127.116.11) are esterases that hydrolyse water-insoluble substrates such as long-chain triglycerides at the interface between the substrate and water. Esterases such as carboxylesterases (EC 18.104.22.168) can also hydrolyze water-soluble esters of carboxylic acids.
 In one embodiment of the invention the polypeptide or a fragment thereof has at least one esterase activity. In another embodiment the activity is a cutinase activity, in still another embodiment the activity is a suberinase activity and in still further embodiment the activity is a lipase activity. In more preferred embodiment the polypeptide or the fragment has two of the above defined activities, most preferably all three of said activities.
 In one embodiment of the invention the polypeptide of the invention or a fragment thereof is active below pH 5; preferably below 4.5, more preferably below 4, below 3.5 or below 3.0, most preferably below 2.5 or even at about 2. Acidic conditions for cutinase treatment are preferable for example in processing food and feed products, since the risk of microbial contamination is reduced at low pH.
 In one embodiment of the invention the polypeptide of the invention or a fragment thereof is active towards polyesters within pH 2.5-7.5; preferably within pH 3.5-6.5. A pH-optimum for cutin hydrolysis is in the range from pH 3.5 to 4.5. The polypeptide also has a local pH-optimum in the range from pH 6 to 7. Between these two optima the activity is also very high, more than 85% of the maximal activity. The pH optimum for pNPB-esterase activity is between 4.5 and 5.
 In one embodiment the polypeptide or fragment thereof is active on fatty acid polyesters.
 Wide pH range for high activity (see Example 6 and FIGS. 3 and 4) makes the enzyme suitable for use under various process conditions, possibly in combination with other enzymes.
 In one embodiment of the invention the polypeptide of the invention or a fragment thereof is stabile within the pH range of 2.0 to 8.0 at 40° C. or at lower temperature. In preferred embodiment the enzyme has 80%, more preferably 90% stability within the pH range of 3.5 to 7.5 at 40° C. or at lower temperature. Wide pH stability is an advantage in processes where the pH conditions are changing during the process. pH properties of the polypeptide of the invention are analyzed in Example 6.
 The polypeptide (or its fragment) of the invention is active at low pH towards substrates of variable molecular masses ranging from polyesters such as cutin or suberin to smaller molecules such as pNPB. This can be a benefit when materials containing different types of esterified substances are hydrolyzed.
 In one embodiment of the invention the polypeptide or a fragment thereof is obtained directly or indirectly from Sirococcus, preferably from Sirococcus conigenus. Discella, Hypoderma, Hysterium and Phoma are synonyms to Sirococcus. Discella strobilina, Hypoderma conigenum, Hysterium conigenum, Phoma conigena, and Sirococcus strobilinus are synonyms to Sirococcus conigenus. In this connection "obtained directly" means that the polypeptide or a fragment is produced by Sirococcus, whereas "obtained indirectly" means that the gene or the sequence information has essentially been derived from Sirococcus. Thus, a synthetic polypeptide having an amino acid sequence of SEQ ID NO: 1 (i.e. sequence of the cutinase polypeptide from Sirococcus conigenus) is within scope of this invention.
 It must be understood that sequence of the gene or a polypeptide obtained from a strain may be modified so that the activity of the encoded polypeptide or fragment is not essentially altered and the same applies to possible post-translational modifications or modifications of synthetic polynucleotide or polypeptide. Typical modification of nucleotides encoding the polypeptide is codon optimization in order to enhance production in foreign production hosts. Examples of post-translational modifications are glycosylation, phosphorylation or digestion to smaller fragments.
 In a preferred embodiment the polypeptide of the invention or a fragment thereof is produced using recombinant technology.
 The invention is also directed to recombinant polynucleotides encoding the polypeptides of the invention. Recombinant polynucleotide is isolated from the genome of the donor organism, or amplified with the genome of the donor organism as the template, or synthesized on the basis of nucleotide data on the genome of the donor organism and transferred to a production organism. It is believed that optimal codons help to achieve faster translation rates and high accuracy in various host organisms, thus, a skilled man understands that also codon optimized sequences are within scope of this invention.
 Polypeptides or fragments of this invention or polynucleotides encoding them can be identified, isolated, cloned and produced by methods known within the art.
 Vectors carrying the nucleotide encoding the polypeptide or a fragment of this invention are also within the scope of this invention. Vectors can be produced by methods known within the art. Typically the expression vector contains means (at least a promoter and a termination signal operably linked to a polypeptide to be produced) for regulating the translation in desired production organism and optionally means for selection and site-directed transformation. A vector can also contain a signal sequence optimal to a production host.
 The vector is then transformed to a host organism, usually a host cell. A stable transformant is preferred and it is also a requirement for large scale production. Any method known to the art may be used. Suitable hosts include fungi, yeasts, bacteria as well as animal and insect cells. Filamentous fungi such as Trichoderma and Aspergillus are examples of suitable fungal hosts, Saccharomyces, Hansenula, Kluyveromyes and Pichia are examples of suitable yeast hosts and Bacillus, Escherichia, Streptomyces, Lactobacillus, Lactococcus and Pseudomonas, are examples of suitable bacterial hosts.
 Host is then cultivated under conditions allowing the expression of the desired polypeptide. The expression level may be increased by known methods such as using multiple copies of the gene to be translated or expressing the gene under a strong promoter such as Trichoderma cbh1 or using a host in which one or more endogeneous genes have been turned on or deleted. E.g. Gellissen (2005) describes recombinant production of polypeptides in various host systems.
 In preferred embodiment the polypeptide (or the fragment) is secreted to the culture medium. The preparation may thus be the spent culture medium containing the polypeptide (usually after the separation of the cells). However, it is also possible to decompose the host cells (if the polypeptide product is not secreted) and thereby release the polypeptide for use or for recovery. The polypeptide can be purified to a higher degree of purity by known methods such us filtration, concentration, affinity methods etc. and formulated to the final product, if needed. In addition, depending on the application it is possible that the preparation also contains the production host.
 In this connection "an enzyme preparation" is any composition comprising the polypeptide(s) or fragment(s) of the invention. Optionally other enzymes and components (such as stabilizers, surface active agents or buffering agents) may be present depending on the field of use. For example, the polypeptide of the invention can be used as a component of a plant material degrading enzyme cocktail containing any of the following enzymatic activities: e.g. protease, cellulase, ligninase, beta-glucosidase, hemicellulase, xylanase, alpha-amylase, amyloglucosidase, pectinases, laccase, peroxidase, lipase or expansin. Furthermore, e.g. protease, alpha-amylase, lipase, cellulase, xylanase, beta-glucanase, pectinase, lipoxygenase, peroxidase or laccase can be used in a detergent composition in combination with the polypeptide of the current invention. The polypeptide in the preparation may be in any form as discussed above.
 In one embodiment of the invention the polypeptide is obtained by transforming a microorganism with a vector carrying the polynucleotide encoding the polypeptide. Stable transformation, e.g. integration of the production construct to the genome of host, is preferred. Host cell is then cultured in conditions that are suitable for expression of the polynucleotide. These conditions greatly depend on type of the host. Preferably the polypeptide is recovered by collecting the culture media where the polypeptide is secreted but also other means are possible.
 Within the scope of this invention is also a method of hydrolysis of ester bonds where material containing ester bonds is contacted with the polypeptide (or a fragment) of this invention under conditions suitable for hydrolysis.
 The method is suitable in releasing cutin or suberin monomers or oligomers from plant materials for the production of cutin or suberin derived chemicals such as polymer additives, polyesters, polyurethans, functionalized polymers, plastisizers, lubricants, compatibilizers, dispertants, base oils, corrosion inhibitors, grease thickeners, biodiesel, alkyd resins, polyurethane resins, drying oils, printing ink additives, wetting agents, viscosity modifiers, emulsifiers, stabilizers, coatings, texturizing additives, antioxidants, dyes, flavor compounds, nutrition supplements, cosmetic products, medicines (e.g. for UV-protection, anti-aging, treating hypercholestemia, preventing mutagenesis and adsorbtion of dietary carcinogens), texturized fats, health beneficial fats, or as reactants of trans- or interesterification reactions.
 One particular embodiment is a method of treating agricultural or food raw materials or by-products obtained for example from vegetables, fruits, grapes, berries or cereals with the polypeptide or the fragment of the invention for facilitating the release of bioactive compounds (for examples polyphenolics) from the native biomatrix or for opening the plant surface structure for other degrading enzymes, for example, cellulases, ligninases, hemicellulases, pectinases or xylanases. In addition, the treatment with the polypeptide or the fragment of the invention may facilitate processing these materials.
 Another embodiment is a method of treating wood raw materials, pulp and paper products, or process wastes or waters, or by-products with said protein for releasing suberin oligomers or monomers and/or for facilitating processing of the wood or wood-derived materials using the polypeptide or the fragment of this invention.
 Another embodiment is a method of modifying materials used in textile production, electronical industry or in biomedicine with said protein in applications in which increasing the hydrophilicity of these materials is required using the polypeptide or the fragment of this invention.
 One embodiment is a method of modifying synthetic or other man-made polyester fibres or textiles using the polypeptide of the fragment of this invention.
 Still further embodiment is a method of removing stickies or fat from laundry and dishes using the polypeptide or the fragment of this invention.
 Another embodiment is a method of releasing flavor-enhancing fatty acids from milk-fat to dairy products using the polypeptide or the fragment of this invention.
 Another embodiment is a method of degrading toxic or harmful ester compounds such as pesticides or plastics using the polypeptide or the fragment of this invention.
 Another embodiment is a method of enhancing the pharmacological effects of chemicals used in agriculture using the polypeptide or the fragment of this invention.
 The various embodiments of this invention, especially the structural and functional properties of the polypeptide having SEQ ID NO: 1 or smaller fragments thereof (smaller polypeptides), can be combined without restriction.
 The above-described embodiments and examples and the attached drawings are given for illustrative purposes and are intended to be non-limiting. The scope of the invention is defined in the following claims which are to be interpreted in their full breadth and taking equivalents into account.
1. Cutinase Assay Using 3H-Labeled Cutin
 Apple cutin was isolated as described previously by Halonen et al. (2009). The extent of cutin hydrolysis was determined using 3H-labeled cutin as the substrate by a modification of a previously described method (Davies et al., 2000). In this method the amount of radioactive hydrolysis products released into the solution from the cutin substrate are measured.
 The labelled cutin (specific activity 4106 dpm/mg) was mixed with unlabelled cutin to achieve a suitable specific activity for radioactivity measurements. 50 μl of enzyme sample was incubated in the presence of 1.2 mg 3H-cutin in a total volume of 200 μl. The reaction mixtures were buffered with McIlvaine. After incubation the reaction mixtures were centrifuged and 150 μl of the supernatants were analyzed using a scintillation counter. All samples were analyzed in duplicates and the average dpm values were calculated. Blank samples with buffer instead of the enzyme solution were used as controls and the values obtained were subtracted from the values obtained with the enzyme samples.
2. p-Nitrophenylbutyrate Esterase Assay
 Esterase activity was determined using p-nitrophenyl butyrate (pNPB) as the model substrate by a modification of the method described previously by Davies et al. (2000). The reaction mixtures (300 μl) contained 2 mM pNPB, 0.5% (w/v) Triton X-100, McIlvaine buffer and 50 μl of the enzyme sample. The change in absorbance was monitored at 340 nm and at 25° C. p-Nitrophenol was used as the standard for the calculation of the initial velocities.
3. Triglyceride Hydrolysis Assay
 Triglyceride hydrolyzing activities of the cutinases were determined using an emulsion of Bertolli olive oil (Unilever) as the substrate essentially as described by Kontkanen et al. (2004). An emulsification reagent was prepared by dissolving 2.5 g of gum arabic from acacia tree into 47% (v/v) glycerol. To 70 ml of the emulsification reagent, 30 ml of olive oil was added and the mixture was homogenized mechanically. The reaction mixtures contained 100 μl of the enzyme sample, 900 μl of McIlvaine buffer, pH 4.5, and 1 ml of the olive oil emulsion. The reaction mixtures were incubated at 40° C. under magnetic stirring for 10 min and the reactions were stopped by placing the tubes in a boiling water bath for 5 min. Then 2 ml of acetone:ethanol (1:1) was added and the phases were allowed to separate. The concentration of free fatty acids was determined from the upper liquid layer using the Free Fatty Acids, Half Micro Test (Roche). All samples were analyzed in duplicates.
4. Determination of Protein Concentrations
 The commercial BioRad DC Protein Assay kit (500-0112) was used according to the instructions by the manufacturer with bovine serum albumin as the standard.
Screening of Microbes for Polyester Hydrolyzing Activity
 55 microbes (mostly fungal strains) from the VTT Culture Collection (Finland) were grown in the presence of suberin in Yeast Peptone or Yeast Nitrogen Base media under slightly acidic conditions and analyzed for suberinolytic activity. Degradation of suberin was analyzed by GC/MS from suberin samples withdrawn from the cultures. For analysis by GC/MS the solid samples were freeze dried. The dry samples were extracted consecutively with hexane and ethanol for lipophilic and hydrophilic compounds, respectively. The extractions were done by Dionex ASE 200 Accelerated Solvent Extraction System. The temperatures used were 90° C. for hexane extractions and 100° C. for ethanol extractions. The pressure was 100 bar in both cases and the extraction time was five minutes.
 The GC-MS-instrument used for the analysis of the extracts for suberin monomers consisted of an Agilent 6890A GC and 5973N MS. The column used was a Nordion NB-54 with 5% phenylmethylpolysiloxane stationary phase. Heptadecanoic acid (100 μg) was added to the samples as an internal standard prior to silylating with N,O-bis(trimethyl-silyl)trifluoroacetamide and trimethylchlorosilane. The temperature program was: 100° C.→15° C.→280° C. (18 min). The data collected was in the mass range from m/z 40-800 amu. Increased amount of long fatty acids, such as hydroxy fatty acids and diols, was used as the indicator for suberin degradation during the cultivations. The ability of the microbes to degrade suberin was also evaluated visually during the cultivations. Furthermore, the growth of the microbes in the absence and presence of suberin was monitored. Sirococcus conigenus was identified as a producer of suberinolytic polyesterases by the methods used.
Cloning and Sequencing of the Sirococcus conigenus Cutinase (ScCut) Gene
 A fragment of the gene encoding the Sirococcus conigenus cutinase (ScCut) enzyme was amplified by PCR (polymerase chain reaction). Genomic DNA of S. conigenus was used as the template. The degenerate primers used were designed on the basis of sequences for fungal cutinases and cutinase-like genes obtained from the GenBank sequence database. These genes were from the fungi Aspergillus oryzae (BAA92327), Pyrenopeziza brassicae (CAB40372) and Botrytis cinerea (XP--001554721). Two different sets of primers were used: in the first set the codon usage of the fungi was not taken into account and in the second set the primers were modified to reflect the codon usage of S. conigenus (Table 1). Dynazyme EXT DNA polymerase (Finnzymes, Finland) was used in the reactions. In the touchdown PCR program used for amplification, the annealing temperature was lowered after every cycle (Table 2). (SEQ ID NO:s 4 to 11)
TABLE-US-00001 TABLE 1 Degenerate primers used for amplification Codon usage taken into Primer Strand account Sequence (5'-3') AsKod1aF coding yes TTCGCYCGYGGYACYTCYGA GCCYGGYAA AsKod1bF coding yes GTCATGTCYGGYTAYTCYCAR GG AsKod2aR non- yes GGRTCRCCGAARATGAC coding AsKod2bR non- yes CCYTGRGARTARCCRGA coding As1aF coding no TTYGCIMGIGGIACIWSI GARCCIGGIAA As1bF coding no GTIATGWSIGGITAYWSI CARGG As2aR non- no GGRTCICCRAAIATIAC coding As2bR non- no CCYTGISWRTAICCISW coding
 In the table I indicates inosine (n in sequence listing), Y is C or T, R is A or G, W is A or T and S is G or C.
TABLE-US-00002 TABLE 2 Touchdown PCR-program for amplification of the fragment of the ScCut gene Step Incubation time and temperature 1 94° C. for 45 seconds 2 50° C. for 1 minutes-1° C./cycle 3 72° C. for 3 minutes 4 14 times back to step 1 5 94° C. for 45 seconds 6 36.9° C. for 1 minute 7 72° C. for 3 minutes 8 25 times back to step 5 9 72° C. for 10 minutes
 A fragment of 329 bp was amplified with the primers AsKodlaF and As2aR. The purified fragment was cloned into the pCR2.1 vector according to the instructions by the manufacturer (Invitrogen). The vector was transformed to Escherichia coli DH5α by electroporation according to the instructions of the TOPO TA-cloning kit manual (Invitrogen). Plasmid DNA was isolated from the transformants using the QIAprep Spin miniprep kit (Qiagen). The fragment cloned to the vector was sequenced using the reagents of the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) according to the instructions by Platt et al. (2007). M13 primers (5'-GTA AAA CGA CGG CCA GT-3' and 5'-CAG GAA ACA GCT ATG AC-3', SEQ ID NO: 12 and SEQ ID NO: 13, respectively) were used for sequencing. According to a blastX search of DNA-databases, the sequence of the fragment was similar to sequences present in several fungal cutinase genes.
 Ligation mediated PCR (Fors et al., 1990) was used for obtaining the whole sequence for the ScCut gene. Genomic DNA was digested with the blunt-end-generating restriction enzymes BmgBI, EcoRV, FspI, NruI and RsaI. The fragments were purified (QiaQuick PCR-purification Kit, Qiagen) and ligated in a linker mixture containing linker I: 5''-G CG GTG ACC CGG GAG ATC TGA ATT C-3' and linker II: 5'-GAA TTC AGA TCT-3' primers (SEQ ID NO: 14 and SEQ ID NO: 15, respectively) with T4 ligase. Ligase and unligated linkers were removed after the reaction using the QiaQuick PCR-purification Kit (Qiagen).
 Four primers were designed (Table 3) on the basis of the sequence of the fragment amplified using the degenerate primers. In the first PCR reactions, the fragments were amplified using the primers Cut2R and linker I (reaction a) and Cut1F and linker I (reaction b). In the second PCR reactions the PCR products from the first reactions were used as the templates. Fragments were amplified using the primer pairs Cut1R and linker I (product from reaction a as the template) and Cut2F and linker I (product from reaction b as the template). Dynazyme EXT DNA polymerase (Finnzymes, Finland) was used in these PCRs and the program used was: 94° C. for 3 minutes followed by 30 cycles of 94° C. for 1 minute, 60° C. for 2 minutes, 72° C. for 2 minutes. The final extension was carried out at 72° C. for 10 minutes. Products from the second PCR reactions were gel purified and sequenced directly with either primer Cut1R (products from the PCR reaction a) or primer Cut2F (products from the PCR reaction b) to obtain the sequence of the ScCut gene. No introns could be identified in the sequence.
TABLE-US-00003 TABLE 3 Primers used for ligation mediated PCR (SEQ ID NO's: 16 to 19). Primer Sequence (5'-3') PCR-reaction Cut1F AATGTTGGCCGGGTAGTCGAC reaction I a Cut1R GGACCATGTCGCAGGTCAACTCG reaction II a Cut2F TTGACAGCAGCGAAGAAGGGC reaction II b Cut2R GCAGCTGGTGCACAACGCGGCCAA reaction I b
 The gene encoding ScCut was amplified using the primers binding upstream of the ScCut gene start codon (SconCutF: 5'-CAG GTC GTA CTG GAT TTC TG-3', SEQ ID NO: 20) and downstream of the stop codon (SconCutR: 5'-ACA GAA GTT TCC TGC CCC TT-3', SEQ ID NO: 21). The PCR program used was: 94° C. for 5 minutes followed by 25 cycles of 94° C. for 45 seconds, 50° C. for 1 minute and 72° C. for 3 minutes. The final extension was carried out at 72° C. for 10 minutes. The Phusion DNA polymerase (Finnzymes, Finland) was used in the reaction. The fragment of the correct size was gel purified and 3'-A overhangs were added to the fragment in a Dynazyme DNA polymerase (Finnzymes, Finland) catalyzed reaction. The fragment was cloned to pCR2.1 vector and the resulting plasmid was transformed into Escherichia coli DH5α. The fragment was sequenced using M13 primers and the primers SconCutF and SconCutR.
 The sequence was then compared to known cutinase sequences (FIG. 1) by ClustalW using default settings and Blosum62 as the substitution matrix (Thompson et al., 1994).
Cloning and Expression of the ScCut Cutinase Gene
 The Sirococcus conigenus cutinase (ScCut) gene (SEQ ID NO: 2) was codon optimized (SEQ ID NO: 3) for expression in Pichia pastoris and synthesized by GenScript (USA). Cloning and expression was carried out essentially as described in the Invitrogen User Manual for Expression of Recombinant Proteins in Pichia pastoris (revision date 7 Sep. 2010, Manual part no. 25-0043). The gene was supplemented with a sequence encoding a C-terminal His-tag to enable one-step purification of the ScCut enzyme by immobilized metal affinity chromatography (IMAC). The original secretion signal sequence of the gene was predicted using the SignalP online tool and omitted from the fragment. To facilitate cloning EcoRI and NotI restriction sites were inserted at the 5' and 3' ends of the fragment, respectively. The gene was isolated from the plasmids sent by GenScript by digestion with EcoRI and NotI and the gene fragment was ligated into the expression vector pPicZa-A (Invitrogen) in which it was fused in frame with the Saccharomyces cerevisiae α-factor secretion signal sequence. The vector contained the strong methanol inducible AOX1 promoter for expression of the gene. The constructs were transformed into Escherichia coli and ten transformant colonies were screened for the correct insert by PCR. Plasmid DNA was isolated from five of the positive clones and the DNAs were analyzed by restriction digestion analysis and by sequencing.
 P. pastoris X-33 was cultivated aerobically in Yeast Extract Peptone Dextrose broth at 30° C. The cells were harvested at the optical density (600 nm) of between 1.3 and 1.5 by centrifugation. The cell pellet was washed with ice cold water and with ice cold 1 M sorbitol. After centrifugation the cells were suspended in 1/125 of the original culture volume in 1 M sorbitol and kept on ice. DNA (7.5 μg) linearized with PmeI digestion was mixed with 80 μl of cell suspension and the cells were transformed by electroporation. 1 ml of 1 M sorbitol was added and the cells were incubated for 1 h at room temperature and plated on Yeast Extract Peptone Dextrose agar plates containing 100 μg/ml of zeocin.
 The transformants were grown overnight in Buffered Glycerol Complex Medium at 30° C. When the optical densities (600 nm) reached 2.5, the cells were collected by centrifugation and suspended into Buffered Methanol Complex Medium containing 0.5% (v/v) methanol to optical density of 0.45 at 600 nm. The cells were grown aerobically at 30° C. for 6 days. Methanol was added each day to 0.5% (v/v) of total volume to compensate for evaporation. Expression of the cutinase genes were followed by measuring the pNPB-esterase activity at pH 3.5 (as described in Example 1) of the growth supernatants. The most efficient esterase producing transformant was chosen for ScCut production.
Production and Purification of the ScCut Enzyme
 1 liter of Buffered Methanol Complex Medium was inoculated with a 50 ml culture of the P. pastoris transformant chosen for cutinase production (described in Example 4) and the cells were grown aerobically at 30° C. for 3 days. pNPB-esterase activity at pH 3.5 (as described in Example 1) was monitored (FIG. 2) and methanol was added daily to 0.5% (v/v) to the culture. After the cultivation the cells were removed by centrifugation and the pH of the supernatant was adjusted to pH 8.0 with NaOH. The supernatant was filtered and 3 g of trisodium citrate-2-hydrate was added to prevent the formation of the phosphate precipitates. 1 mM of the protease inhibitor phenylmethanesulfonyl fluoride was added to the supernatant.
 The C-terminal His-tag with 6 histidines fused to the ScCut enabled the one step purification of the enzyme by IMAC. Ni-NTA-Agarose (Qiagen, 30210) packed in a XKl6 column (Pharmasia) with a column volume of 4 ml was used in the purification. The column was equilibrated with starting buffer containing 50 mM Na-phosphate, pH 8.0, and 300 mM of NaCl. The sample (850 ml) was applied at a flow rate of 1 ml/min. The column was eluted with starting buffer supplemented with 10 mM of imidazole and fractions having pNPB-esterase activity at pH 3.5 (determined as described in Example 1) were collected. The buffer of these fractions was changed to 1:10 McIlvaine, pH 7, by ultrafiltration and dilution. SDS-PAGE analysis of the ScCut containing fractions showed a protein band with a molecular mass of around 20 kDa. This corresponds well with the molecular mass of 18.2 kDa calculated on the basis of the amino-acid sequence. The ScCut was purified to a virtual homogeneity by IMAC. The purified protein had a specific pNPB-esterase activity of 360 nkat/mg at pH 3.5. The purified sample was used for further enzyme characterization experiments.
pH-Profile and -Stability of ScCut
 The pH-profile of ScCut was studied between pH 2 and 8 for cutinolytic activity (using 3H-cutin as the substrate) and for pNPB-esterase activity. The assay procedures described in Example 1 were used in the experiments. The reaction mixtures were buffered with McIlvaine buffer. The pH profile for cutin hydrolysis by ScCut was determined by incubating the enzyme samples at 40° C. for 24 h at a protein dilution of 0.2 mg/ml. The pNPB-esterase activities were determined at 25° C. using a protein dilution of 7.4 μg/ml. The results are presented in FIG. 3. With 3H-cutin as the substrate the values are presented as percentages of the maximum. ScCut showed a pH-optimum for cutin hydrolysis between 3.5 and 4.5, whereas the pH optimum for pNPB-esterase activity was between 4.5 and 5. With both 3H-cutin and pNPB as the substrates, hydrolysis was detected across a broad pH range. Activity was detectable below pH 3 with both substrates. The cutin hydrolyzing capacity of ScCut was compared to the results reported for the most acidic cutinases known [Botrytis cinerea cutinases (Shishiyama et al., 1970; Salinas et al., 1986; van der Vlugt-Bergmans et al., 1997), Venturia inaequalis cutinase (Koller and Parker, 1989) and Alternaria brassicola cutinase (Trail and Koller, 1993)]. It can be concluded on the basis of the comparison that cutin hydrolysis at pHs of below 5.0 has not been shown for any other cutinase than ScCut.
 pH stability was determined at the same pH range as above by incubating the enzyme samples at 40° C. for 24 h in McIlvaine buffer. After incubation the samples were diluted by 1:5 with McIlvaine buffer at pH 7 and the pNPB-esterase activities were determined. The reaction mixtures were buffered with 0.5 M Na-phosphate buffer pH 7 in order to bring all samples to equal pH. The residual pNPB-esterase activities were determined and compared to the initial activity determined at pH 7.0. The results are presented in FIG. 4. ScCut was stable over a wide range of pH-values. It showed over 90% stability within the pH range of 3.5-7.5. The stability was somewhat lower outside this range, but clear residual activity was detected. The results show that the enzyme is both stable and active under various pHs and could be suitable for many different applications because of this type of robustness.
Temperature Stability of ScCut
 The temperature stability of ScCut was studied by determining the half lives at different temperatures. ScCut was incubated at the concentration of 37 μg/ml in McIlvaine buffer at pH 4.5. pNPB-esterase activities were measured from each sample at multiple time points during two days. The results were plotted on a semi-log scale (log of the relative activity versus time), and the half-lives were calculated from the slope of the trend line. No decrease in the activity was detected at 4° C. and at 25° C. during the two-day incubation. The half-lives determined at the other temperatures used are presented in Table 4. The results show that ScCut is stable for use in detergent and plant material processing applications.
TABLE-US-00004 TABLE 4 Half-lives of ScCut at different temperatures Temperature Half-life 42° C. 76 h 55° C. 9 h 65° C. 4 h 85° C. 24 min
Hydrolysis of p-Nitrophenyl Esters of Different Chain Lengths
 The activity of ScCut towards fatty acid esters with different carbon chain lengths was determined at pH 4.5 using the procedure for the esterase assay for pNP-butyrate hydrolysis (described in Example 1). The fatty acid moieties of the p-nitrophenyl esters used as the substrates were: acetate (C2), propionate (C3), butyrate (C4), valerate (C5), caproate (C6), caprate (C10), laurate (C12), myristate (C14), palmitate (C16) and stearate (C18). The substrate concentration of 2 mM was used in the reaction mixtures. The results shown in FIG. 5 indicate a preference towards pNP-esters of short chain fatty acids by ScCut. This specificity profile is typical for cutinases.
Hydrolysis of Cutin, Suberin and Triglycerides by ScCut
 The natural, extractive free substrates, apple cutin and birch bark suberin were isolated as described previously by Halonen et al. (2009) for estimation on ScCut catalyzed hydrolysis of these materials. The reaction mixtures comprised 50 mg/ml of cutin or suberin and 100 nkat/ml of ScCut (as pNPB-esterase activity determined at pH 4.5) and McIlvaine buffer at pH 4.5. The reaction mixtures were incubated for 48 h at 40° C. and extracted with methyl tertiary butyl ether (MTBE) in order to recover the cutin and suberin oligo- and monomers released from the solid substrate by enzyme action. Free fatty acids in the MTBE extract were analysed directly and after alkali hydrolysis of released oligomers using the Free Fatty Acids Half Micro Test (Roche) as described previously by Kontkanen et al. (2009). The amounts of fatty acids released by ScCut treatment were compared to the amount of fatty acids released in total alkali hydrolysis of the substrates. Treatment with ScCut released 1.4 mol-% and 3.0 mol-% of the total fatty acids from suberin and cutin, respectively.
 The specific lipase (triglyceride hydrolyzing) activity was determined at pH 4.5 with olive oil as the substrate as described in Example 1. ScCut showed a specific lipase activity of 70.3±3.7 nkat/mg.
 Agrawal P. B. (2005) The performance of cutinase and pectinase in cotton scouring. PhD Thesis, University of Twente, the Netherlands.
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211202PRTSirococcus conigenusSIGNAL(1)..(17)mat_peptide(18)..(202) 1Met Lys Ser Thr Ile Leu Phe Ser Ala Leu Leu Ser Ser Gly Ala Leu -15 -10 -5 Ala Ala Pro Thr Val Val Glu Ala Val Glu Ala Arg Ala Ala Cys Ser -1 1 5 10 15 Asp Met Thr Ile Ile Phe Ala Arg Gly Thr Thr Glu Pro Gly Thr Val 20 25 30 Gly Thr Leu Ala Gly Pro Pro Phe Phe Ala Ala Val Lys Ser Gln Leu 35 40 45 Gly Gly Arg Ala Thr Leu Thr Thr Gln Gly Val Asp Tyr Pro Ala Asn 50 55 60 Ile Ala Gly Phe Leu Ala Gly Gly Asp Pro Ala Gly Ser Gln Thr Met 65 70 75 Ala Asn Asp Val Lys Ala Ala Leu Ala Ala Cys Pro Asp Thr Lys Leu 80 85 90 95 Val Met Ala Gly Tyr Ser Gln Gly Gly Gln Leu Val His Asn Ala Ala 100 105 110 Lys Leu Leu Gly Gly Thr Met Ser Gln Val Asn Ser Ala Val Ile Phe 115 120 125 Gly Asp Pro Asp Asn Gly Gln Pro Val Ala Gly Leu Ser Ala Ala Gln 130 135 140 Thr Lys Ile Ile Cys His Ala Gly Asp Asn Ile Cys Gln Gly Gly Ala 145 150 155 Leu Ile Leu Ala Pro His Leu Thr Tyr Gly Gln Asp Ala Gly Thr Ala 160 165 170 175 Ala Ser Phe Val Ile Ala Ala Ala Gly Leu 180 185 2609DNASirococcus conigenus 2atgaagtcta ccattctctt ttcggccctc ctctcatcgg gtgctctggc agcacctacc 60gttgtcgagg ccgttgaagc acgcgccgcc tgctcggaca tgaccattat ctttgcccgc 120ggcaccaccg agccgggcac cgtcggcacc ctggctggtc cgcccttctt cgctgctgtc 180aagtcccagt tgggcgggag ggccacgctg accacccagg gcgtcgacta cccggccaac 240attgccggct tcctggccgg cggcgacccg gccgggagcc agaccatggc caacgacgtc 300aaggcggcgc tggcggcgtg ccccgacacc aagctggtca tggcgggcta ctcgcagggc 360gggcagctgg tgcacaacgc ggccaagctc ttggggggga ccatgtcgca ggtcaactcg 420gccgtcatct ttggcgaccc ggacaacggc cagcccgtgg cgggcctgtc ggcggcccag 480acgaaaatca tctgccacgc cggcgacaac atttgccagg gaggcgccct gatcctggcg 540ccgcacctga cgtacgggca ggacgcgggc actgcagcct cttttgtgat cgcagcggcc 600gggctttaa 6093590DNASirococcus conigenus 3gaattcgctc caaccgttgt cgaagcagtt gaggctagag ctgcctgttc tgacatgact 60attatctttg ccagaggtac tacagaacct ggtacagttg gaaccttggc aggaccacca 120tttttcgcag ctgtcaagtc tcaattgggt ggtagagcta ctcttaccac tcagggagtt 180gattacccag ccaacattgc aggtttcttg gcaggtggag atcctgctgg atcacaaaca 240atggctaatg acgtcaaggc cgcattggct gcctgcccag ataccaaact tgttatggct 300ggttatagtc aaggtggaca gttggtccat aacgctgcta agttgcttgg tggaactatg 360tctcaagtta actccgccgt catttttgga gatccagaca atggacaacc tgttgctggt 420ttgtctgccg cacagacaaa aattatctgt catgctggtg acaatatttg ccaaggtgga 480gctttgatcc ttgcccctca cttgacttac ggtcaggatg ctggaacagc tgcctctttc 540gttatcgcag ctgccggtct tcatcaccat caccatcact aagcggccgc 590429DNAArtificial SequencePrimer 4ttcgcycgyg gyacytcyga gccyggyaa 29523DNAArtificial Sequenceprimer 5gtcatgtcyg gytaytcyca rgg 23617DNAArtificial Sequenceprimer 6ggrtcrccga aratgac 17717DNAArtificial Sequenceprimer 7ccytgrgart arccrga 17829DNAArtificial Sequenceprimer 8ttygcnmgng gnacnwsnga rccnggnaa 29923DNAArtificial Sequenceprimer 9gtnatgwsng gntaywsnca rgg 231017DNAArtificial Sequenceprimer 10ggrtcnccra anatnac 171117DNAArtificial Sequenceprimer 11ccytgnswrt anccnsw 171217DNAArtificial Sequenceprimer 12gtaaaacgac ggccagt 171317DNAArtificial Sequenceprimer 13caggaaacag ctatgac 171425DNAArtificial Sequenceprimer 14gcggtgaccc gggagatctg aattc 251512DNAArtificial Sequenceprimer 15gaattcagat ct 121621DNAArtificial Sequenceprimer 16aatgttggcc gggtagtcga c 211723DNAArtificial Sequenceprimer 17ggaccatgtc gcaggtcaac tcg 231821DNAArtificial Sequenceprimer 18ttgacagcag cgaagaaggg c 211924DNAArtificial Sequenceprimer 19gcagctggtg cacaacgcgg ccaa 242020DNAArtificial Sequenceprimer 20caggtcgtac tggatttctg 202120DNAArtificial Sequenceprimer 21acagaagttt cctgcccctt 20
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